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The efficacy of cell-based therapies as an alternative to autologous bone grafts requires biomaterials to localize cells at the defect and drive osteogenic differentiation. Hydrogels are ideal cell delivery vehicles that can provide instructional cues via their composition or mechanical properties but commonly lack osteoconductive components that nucleate mineral. To address this challenge, we entrapped mesenchymal stromal cells (MSCs) in a composite hydrogel based on two naturally-derived polymers (alginate and hyaluronate) containing biomineralized polymeric microspheres. Mechanical properties of the hydrogels were dependent upon composition. The presentation of the adhesive tripeptide Arginine-Glycine-Aspartic Acid (RGD) from both polymers induced greater osteogenic differentiation of ovine MSCs in vitro compared to gels formed of RGD-alginate or RGD-alginate/hyaluronate alone. We then evaluated the capacity of this construct to stimulate bone healing when transplanting autologous, culture-expanded MSCs into a surgical induced, critical-sized ovine iliac crest bone defect. At 12 weeks post-implantation, defects treated with MSCs transplanted in composite gels exhibited significant increases in blood vessel density, osteoid formation, and bone formation compared to acellular gels or untreated defects. These findings demonstrate the capacity of osteoconductive hydrogels to promote bone formation with autologous MSCs in a large animal bone defect model and provide a promising vehicle for cell-based therapies of bone healing.

Recombinant proteins and cytokines are under broad preclinical and clinical investigation to promote angiogenesis, but their success is limited by ineffective delivery, lack of long-term stability and excessive cost. Mesenchymal stem/stromal cells (MSC) secrete bioactive trophic factors, and thus, may provide an effective alternative to address these challenges. Glycine-Histidine-Lysine (GHK) is a peptide fragment of osteonectin, a matricellular protein with reported proangiogenic potential. We examined the capacity of GHK to up-regulate secretion of proangiogenic factors from human MSC in culture and when covalently coupled to alginate hydrogels. GHK had no apparent cytotoxic effects on MSC in culture over a wide range of concentrations. We detected a dose-dependent increase in vascular endothelial growth factor (VEGF) concentration in media conditioned by GHK-treated MSC, which increased endothelial cell proliferation, migration and tubule formation. We covalently coupled GHK to alginate using carbodiimide chemistry, and human MSC were entrapped in alginate hydrogels to assess VEGF secretion. Similar to monolayer culture, MSC responded to GHK-modified gels by secreting increased concentrations of VEGF and basic fibroblast growth factor compared to unmodified gels. The pre-treatment of MSC with antibodies to α6 and β1 integrins prior to entrapment in GHK-modified gels abrogated VEGF secretion, suggesting that the proangiogenic response of MSC was integrin-mediated. These data demonstrate that the proangiogenic potential of MSC can be significantly increased by the presentation of GHK with a biodegradable carrier, therefore increasing their clinical potential when used for tissue repair.

The increasing prevalence of bone‐related diseases has raised concern about the need for an osteoinductive and mechanically stronger scaffold‐based bone tissue engineering (BTE) alternative. A mineralized microenvironment, similar to the native bone microenvironment, is required in the scaffold to recruit and differentiate local mesenchymal stem cells at the bone defect site. Further, extracellular vesicles (EVs), pre‐osteoblasts' secretome, contain osteoinductive cargo and have recently been exploited in bone regeneration. This work developed a cell‐free and mechanically strong interpenetrating network‐based scaffold for BTE by combining the action of osteoinductive EVs with a mineralized microenvironment. The MC3T3 (a pre‐osteoblast cell line) is used as a source of EVs and as the target population. The optimal concentration of MC3T3‐EVs was first determined to induce osteogenesis in target cells. The osteoinductive potential of the scaffold was estimated in vitro by osteogenesis‐related markers like the alkaline phosphatase (ALP) enzyme and calcium content. The MC3T3‐EVs cargo was also studied for osteoinductive signals such as ALP, calcium, and mRNA. The findings of this work indicated that MC3T3‐EVs at a 90 μg/mL dose had significantly higher ALP activity than 0 μg/mL (1.47‐fold), 10 μg/mL (1.41‐fold), and 30 μg/mL (1.39‐fold) EV‐concentration on day 14. Further combination of the optimum dose of EVs with a mineralized microenvironment significantly enhanced ALP activity (1.5‐fold) and mineralization (3.36‐fold) as compared to the control group on day 7. EV cargo analysis revealed the presence of calcium, the ALP enzyme, and the mRNAs necessary for osteogenesis and angiogenesis. ALP activity was significantly boosted in the EV‐containing target cells as early as day 1, and mineralization began on day 7 because MC3T3‐EVs carry ALP enzymes and calcium as cargo. When osteoinductive EVs were combined with an osteoconductive mineralized microenvironment, osteogenesis was significantly enhanced in target cells at early time points. The interaction between osteoinductive EVs and the mineralized milieu facilitates the process of osteogenesis in the target cells and suggests a potential cell‐free strategy for in vivo bone repair. [ABSTRACT FROM AUTHOR]

The skin is one of the most essential tissues in the human body, interacting with the outside environment and shielding the body from diseases and excessive water loss. Hydrogels, decellularized porcine dermal matrix, and lyophilized polymer scaffolds have all been used in studies of skin wound repair, wound dressing, and skin tissue engineering, however, these materials cannot replicate the nanofibrous architecture of the skin's native extracellular matrix (ECM). Electrospun nanofibers are a fascinating new form of nanomaterials with tremendous potential across a broad spectrum of applications in the biomedical field, including wound dressings, wound healing scaffolds, regenerative medicine, bioengineering of skin tissue, and multifaceted drug delivery. This article reviews recent in vitro and in vivo developments in multifunctional electrospun nanofibers (MENs) for wound healing. This review begins with an introduction to the electrospinning process, its principle, and the processing parameters which have a significant impact on the nanofiber properties. It then discusses the various geometries and advantages of MEN scaffolds produced by different innovative electrospinning techniques for wound healing applications when used in combination with stem cells. This review also discusses some of the possible future nanofiber‐based models that could be used. Finally, we conclude with potential perspectives and conclusions in this area. [ABSTRACT FROM AUTHOR]

Designing a temporary substrate to adhere, multiply and form new bone tissue for the bone-forming cells is a challenge in bone tissue engineering. In this article, a new biodegradable and composite scaffolding system was fabricated using an electrospinning technique to augment bone formation using poly-caprolactone (PCL), natural polymer gelatin (GL) and nano-hydroxyapatite (nHAp). The effect of fabrication parameters on nanofiber scaffolds (PCL, PCL + GL, PCL + GL + nHAp) production, as well as MC3T3-E1 proliferation and osteogenic differentiation was investigated. Physical properties of scaffolds, such as morphology, surface area, wettability, were examined using SEM, BET method, and contact angle measurements, respectively. A pre-osteoblastic cell line, MC3T3-E1 was seeded on these scaffolds to study the proliferation by DNA assay and cell viability using live-dead assay. After 21 days of in vitro culture, more than 89% of the MC3T3 cells remained viable within PCL + GL + nHAp scaffold, in contrast to 61.9% viability observed in the plain PCL scaffold. The differentiation of MC3T3 cells into the bone phenotype was determined quantitatively using alkaline phosphatase (ALP) activity and calcium deposition as well as qualitatively using alizarin red staining. At day 21, the DNA content of PCL + GL + nHAp scaffold was 4.5 times higher than its day 1 DNA contents. At day 21, the normalized intracellular ALP activity in the PCL + GL + nHAp group was 1.2 times higher than that in the PCL + GL group, which in turn was 1.7 times larger than that in the PCL group. Moreover, at day 21, PCL + GL + nHAp scaffold showed a 12.43-fold increase in ALP activity and deposited nearly 35-fold higher calcium relative to their respective values at day 1, thus indicating that the inclusion of gelatin and nHAp significantly enhanced cell binding, long-term cell survivability, proliferation, and stimulated osteogenesis in vitro. The findings from current work show that incorporation of nHAp into the nanofibrous scaffold of PCL + GL and the use of our optimized electrospinning process provides a favorable substrate to promote bone healing. [ABSTRACT FROM AUTHOR]

Osteo-odonto-keratoprostheses, incorporating dental laminate material as an anchoring skirt around a central poly(methyl methacrylate) (PMMA) optic, have been used to replace the cornea for many years. However, there are many intricacies associated with the use of autologous dental laminate material, surgical complexity and skirt erosion. Tissue engineering approaches to bone replacement may offer suitable alternatives in osteo-odonto-keratoprosthesis (OOKP) surgery. In this study, a hydrogel polymer composite was investigated as a synthetic substitute for the OOKP skirt. A novel high strength interpenetrating network (IPN) hydrogel composite with nano-crystalline hydroxyapatite (nHAp) coated poly (lactic-co-glycolic acid) PLGA microspheres was created to mimic the alveo-dental lamina by employing agarose and poly(ethylene glycol) diacrylate (PEGDA) polymers. The incorporation of nHAp coated PLGA microspheres into the hybrid IPN network provide a micro-environment similar to that of skeletal tissues and improve cellular response. Agarose was used as a first network to encapsulate keratocytes/3T3 fibroblasts and PEGDA (6000 Da) was used as a second network with varying concentrations (20 and 40 wt %) to produce a strong and biocompatible scaffold. An increased concentration of either agarose or PEG-DA and incorporation of nHAp coated PLGA microspheres led to an increase in the elastic modulus. The IPN hydrogel combinations supported the adhesion and proliferation of both fibroblast and ocular human keratocyte cell types during in in-vitro testing. The cells endured the encapsulation process into the IPN and remained viable at 1 week post-encapsulation in the presence of nHAp coated microspheres. The material did not induce significant production of inflammatory cytokine IL-6 in comparison to a positive control (p < 0.05) indicating non-inflammatory potential. The nHAp encapsulated composite IPN hydrogels are mechanically strong, cell supportive, non-inflammatory materials supporting their development as OOKP skirt substitutes using a new approach to dental laminate biomimicry in the OOKP skirt material. [ABSTRACT FROM AUTHOR]